Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture

Preparation of human platelet lysate (HPL)

HPL in this study was prepared from leukocyte-poor platelet concentrate (LPPC) collected from the hospital blood bank. Only the group “O” pooled LPPCs with “out of date” labelling (five-day shelf life) were employed in our experiment. Platelet counts were performed by manual cell counting and the range of platelet number per LPPC unit was 7 × 10⁸ cells/ml to 8 × 10⁸ cells/ml before storage in a freezer at -80°C until use. HPL was prepared by freezing and thawing. The outdated LPPC that was stored in a freezer at -80°C was immersed in a water bath at 37°C for one hour to thaw. After thawing, the lysed platelet was centrifuged at 4000 g, 4°C for 20 minutes to remove cell debris. The supernatant was collected and filtered with a 40 µm cell strainer. The HPL was stored at 2°C to 8°C for up to four weeks. For medium preparation, 2 ml heparin was added to the medium to prevent clot formation. The complete medium was filtered with a 0.2 µm filter before use.

Preparation of human plasma (Hplasma)

Hplasma in this study was prepared from FFP collected from the hospital blood bank. Only the group “AB” FFP with “out of date” labelling (one-year shelf life) was employed in our experiment. Five units of the outdated FFP were pooled, transferred to centrifuge tubes and stored in a freezer at -20°C until use. To prepare Hplasma, the FFP was thawed at 37°C in a water bath until completely dissolved. To prevent clot formation during culture, 20% (W/V) calcium chloride was added to the FFP at a ratio of 1:100, and FFP was then incubated at 37°C in a water bath for two hours to allow clot formation. After incubation, the fibrin clot was removed by centrifugation at 4000 g, 4°C for 20 minutes. The supernatant was collected, filtered with a 40 µm cell strainer, and stored at 2°C to 4°C for up to four weeks.

Isolation and culture of ADMSCs

ADMSCs were obtained from lipoaspiration of healthy donors. Ethical approval for the collection of tissue and subsequent research was granted by the Committee on Human Rights Related to Research Involving Human Subjects (number MURA2015/149). All subjects were informed and signed a consent form. The lipoaspirate fraction was washed several times with phosphate buffer saline (PBS) and digested with 1 mg/ml collagenase type II (Sigma-Aldrich, St Louis, Missouri) for one hour at 37°C in a shaking water bath. After digestion, the stromal vascular fraction (SVF) was obtained by centrifugation at 2500 g for ten minutes. The SVF portion was collected and washed twice with basal medium. After centrifugation, the pellet was resuspended in complete medium, which was composed of Dulbecco's Modified Eagle Medium (DMEM), 10% FBS (Merck Millipore, Temecula, California), 1X GlutaMAX (Gibco; Thermo Fisher Scientific, Waltham, Massachusetts), 100 U/ml penicillin and 100 µg/ml streptomycin, followed by plating in a T75 cell culture flask. All flasks were cultivated in a humidified incubator at 37°C with 5% CO₂. Non-adherent cells were removed after cultivation for 24 hours. The resulting plastic-adherent cell population was termed ADMSCs.

All of the experiments were performed using ADMSCs prepared according to the procedure that used FBS for the initial ADMSC isolation and expansion. Nevertheless, the collection and expansion of ADMSCs using HPL and Hplasma supplement can be done from an early stage of ADMSC production (supplementary material).

Cell growth

To determine the cell growth potential of MSCs, the periodic monitoring of cell numbers using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and population doubling time (PDT) was employed in this study. For the MTT assay, an indirect measurement of cell number is made by assessing mitochondrial activity of metabolically intact cells. Cells were seeded in 96-well plates at a density of 1×10³ cells per well in 150 µl complete medium containing different supplements. On each day (day 0 to 8) of the MTT assay, 50 µl of 1 mg/ml MTT reagent (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts) was added to each well and the plate was incubated for four hours at 37°C with light protection. The supernatant was then carefully discarded and 100 µl dimethyl sulfoxide (DMSO) was added to each well to solubilise the formazan crystals. The plates were agitated for 15 minutes to dissolve the crystals completely, and the absorbance was measured at 570 nm using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, Vermont).

PDT of the ADMSCs was assessed at passages P4-6. The cells were seeded at 1×10⁵ cells per T25 culture flask with different supplements. After reaching 80% to 90% confluence, cells were trypsinised and counted. The PDT of ADMSCs cultured with different supplements was calculated according to the following formula: PDT = (t × log 2)/(log Nh – log Ni), where t = culture time (h), Nh = number of harvesting cells, and Ni = number of initiating cells.²⁰

Colony-forming unit – fibroblast (CFU-F) assay

The colony-forming capacity of ADMSCs cultured in FBS, HPL, and Hplasma was compared. Cells at passages four to seven were seeded at 100 cells per well in six-well plates in a medium containing different supplements, and cultured for 11 days. Colonies were fixed with methanol for five minutes, stained with 0.5% crystal violet solution for 30 minutes at room temperature, and washed with distilled water to remove excess dye. The colonies, which were defined as a group of more than 50 cells, were counted manually under an inverted microscope.

Immunophenotyping

The expression of ADMSC surface markers was assessed by flow cytometric analysis. Cells were trypsinised into a single cell suspension and incubated with the fluorochrome-conjugated antibodies against human antigens, including anti-CD34-PE, anti-CD45-PerCP, anti-CD73-PE/Cy7, anti-CD90-APC, and CD105-PE (BD Biosciences, San Jose, California), at 4°C for 30 minutes. The cells were then washed twice with cold PBS and fixed with 1% paraformaldehyde before analysis by FACSCanto II Flow Cytometer (BD Biosciences) using FACSDiva software.

Osteogenic and adipogenic differentiation

For osteogenic differentiation, 1×10⁵ ADMSCs were seeded in a 35 mm dish and cultured in an osteogenic differentiation medium composed of DMEM, 10% FBS, 100 nM dexamethasone (Sigma-Aldrich), and 50 µg/ml ascorbic acid (Sigma-Aldrich). After the first week of culture, 10 mM β-glycerophosphate (Merck Millipore) was added to the medium. Cells were cultured for three weeks. To determine bone matrix mineralisation, cells were fixed with 10% formaldehyde for ten minutes and stained with 40 mM Alizarin Red S (Merck Millipore) for 20 minutes at room temperature. For adipogenic differentiation, 1×10⁵ ADMSCs were seeded in a 35 mm dish and cultured in an adipogenic differentiation medium composed of DMEM, 10% FBS, 500 µM isobutyl methylxanthine, 1 µM dexamethasone, 10 µM insulin and 200 µM indomethacin (Sigma-Aldrich). Cells were cultured for two weeks. Adipogenic differentiation was detected by staining of lipid droplets with Oil Red O (Sigma-Aldrich). Cells were washed with PBS, fixed with formalin vapour for ten minutes, and stained with 0.5% Oil Red O in isopropanol for 20 minutes.

Senescence assay

Cellular senescence of ADMSCs was determined at the seventh passage of ADMSCs which had been cultured in medium containing different supplements including FBS, HPL, Hplasma, and HPL+Hplasma. Cells were seeded in a 35 mm dish at a density of 2.5×10⁴ cells per dish in 2 ml of each medium and allowed to grow for three days. Cellular senescence was detected using a senescence-associated β-galactosidase (SA-β-gal) staining kit (Cell Signaling Technology, Inc., Danvers, Massachusetts) according to the manufacturer’s instructions. Briefly, cells were washed twice with PBS, fixed with 1x fixative solution for 15 minutes, and stained with the freshly prepared β-gal staining solution at a pH of 5.9 to 6.1. Dishes were sealed with parafilm to prevent evaporation and incubated at 37°C overnight in the absence of carbon dioxide. The presence of a blue colour, i.e. senescent cells, was observed under an inverted microscope. Ten fields of 10× magnification per dish were taken, and senescent cells were manually counted in each field. Data were presented as a percentage of senescent cells over the total number of cells.

Statistical analysis

Data were presented as mean and standard error of the mean (sem). Comparisons were analysed using the Kruskal-Wallis test followed by Dunn's multiple comparison test, and p < 0.05 was considered to be statistically significant. Statistical tests were performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, California).

Effect of human supplements on morphology and growth of ADMSCs

ADMSCs were cultured in medium containing different supplements: 10% FBS; 10% HPL; 10% Hplasma; and 5% HPL + 5% Hplasma. The morphology of ADMSCs from all conditions were spindle-shaped, fibroblast-like cells. However, ADMSCs cultured in HPL and/or Hplasma were more spindle-shaped and slightly smaller than those cultured in FBS (Fig. 1a). These characteristics could be observed throughout the long-term culture.

Fig.

Morphology and growth of adipose-derived mesenchymal stem cells (ADMSCs) after culture in different supplements: a) ADMSCs showed fibroblast-like morphology after culture in each condition; b) the growth rates of ADMSCs after culture in different supplements for two, four, six, and eight days were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The growth of ADMSCs cultured in human platelet lysate (HPL) and HPL+human plasma (Hplasma) was significantly greater than that of those in fetal bovine serum (FBS). Data were presented as mean and standard error of the mean. * p ⩽ 0.05 compared with FBS. Statistical analysis was tested by Kruskal-Wallis test followed by Dunn's multiple comparison test.

The growth of ADMSCs from these conditions was compared using MTT assay and PDT. As the MTT results show, HPL and HPL+Hplasma exhibited more stimulation of cell growth compared with FBS and Hplasma alone. Cell growth was significantly higher in HPL compared with FBS on day eight of the MTT assay. The growth of ADMSCs in Hplasma was comparable with that in FBS (Fig. 1b). Population doubling time was calculated from three passages of ADMSCs cultured in each group. The mean PDTs of ADMSCs in FBS, HPL, Hplasma, and HPL+Hplasma were 132 (sem 8), 50 (sem 1), 65 (sem 8) and 65 hours (sem 4), respectively (n = 3). The ADMSCs cultured in HPL, Hplasma, and HPL+Hplasma had faster growth kinetics than those cultured in FBS. The PDT was significantly lower in HPL conditions compared with FBS conditions (p < 0.05, Kruskal-Wallis test followed by Dunn's multiple comparison test).

Human supplements maintain typical characteristics of ADMSCs

The influence of different supplements in the maintenance of MSC characteristics was examined. ADMSCs were cultured with different supplements for several passages until reaching the tenth passage, when they were harvested for investigation of several characteristics of MSCs. ADMSCs cultured in HPL and/or Hplasma expressed typical MSC surface markers (similar to FBS conditions): CD73; CD90; and CD105, but not haematopoietic markers; CD34 and CD45.(Fig. 2a, Table I). The differentiation properties of ADMSCs cultured in each supplement were determined by inducing differentiation towards osteogenic and adipogenic lineages. Osteogenic differentiation was indicated by formation of bone matrix mineralisation which was demonstrated by Alizarin Red S staining (Fig. 2b). Adipogenic differentiation was indicated by detection of lipid droplets in the cells with Oil Red O staining (Fig. 2c). Results showed that ADMSCs cultured in human supplements were able to differentiate into both osteogenic and adipogenic lineages similar to those cultured in FBS.

Fig.

Characterisation of Adipose-derived mesenchymal stem cells (ADMSCs) after long-term culture in different supplements: a) immunophenotype of ADMSCs was determined by flow cytometry; b) osteogenic and adipogenic differentiation of ADMSCs was assessed by Alizarin Red S and Oil Red O staining, respectively; c) macroscopic examination of osteogenic differentiation which was assessed by Alizarin Red S staining at days 18, 24, and 30 (FBS, fetal bovine serum; HPL, human platelet lysate; Hplasma, human plasma).

To compare the osteogenic differentiation potential of ADMSCs in each supplement, ADMSCs during osteogenic differentiation at days 18, 24 and 30 were stained by Alizarin Red S. ADMSCs cultured in HPL and/or Hplasma exhibited more robust osteogenic differentiation compared with those cultured in FBS (Fig. 2c).

Effect of human supplements on self-renewal property of ADMSCs

The self-renewal property of ADMSCs was determined by CFU-F assays that were performed in four consecutive passages after culture in each condition. Variation in size and cell density of CFU-F generated from ADMSCs of different conditions was observed. ADMSCs in HPL and HPL+Hplasma condition generated similar characteristics of CFU-F which were larger and denser than those of Hplasma or FBS (Fig. 3a). Although the number of CFU-Fs declined with further cell passages, ADMSCs in HPL, Hplasma, and HPL+Hplasma conditions generated more CFU-Fs than FBS, indicating that human supplements could efficiently maintain the self-renewal property of ADMSCs after long-term culture (Fig. 3b).

Fig.

Colony-forming unit – fibroblast (CFU-F) assay of adipose-derived mesenchymal stem cells (ADMSCs) after culture in different supplements: a) macroscopic (upper panel) and microscopic (lower panel) examination of CFU-F revealed the variation in size and cell density of colonies generated from ADMSCs of different culture conditions. The large and dense colonies were frequently observed in ADMSCs of human platelet lysate (HPL) and HPL+human plasma (Hplasma) condition; b) number of CFU-Fs generated from ADMSCs at passages four to seven was counted and presented as CFU per 100 cell seeding. Data were presented as a mean generated from three independent experiments (FBS, fetal bovine serum).

Human supplements prevent cellular senescence of ADMSCs

The effect of different supplements on cellular senescence of ADMSCs was evaluated by the detection of senescence-associated β-galactosidase (SA-β-gal). ADMSCs were cultured with different supplements for several passages until reaching the seventh passage. These cells were stained with SA-β-gal and the senescent cells appeared as a blue colour that could be observed under an inverted microscope (Fig. 4a). As revealed by the quantitative analysis, the number of senescent cells in ADMSCs cultured in HPL, Hplasma, and HPL+Hplasma was lower than that cultured in FBS (Fig. 4b). Thus, human supplements could prevent cellular senescence of ADMSCs after long-term culture.

Fig.

Cellular senescence assay of adipose-derived mesenchymal stem cells (ADMSCs) after long-term culture in different supplements: a) cellular senescence of ADMSCs was determined by SA-β-gal staining. The senescent cells appeared as a blue colour (arrow); b) senescent cells were counted and presented as a percentage of total cell number. The graph represents mean and standard error of the mean. * p ⩽ 0.05; statistical analysis was tested by Kruskal-Wallis test followed by Dunn's multiple comparison test. (FBS, fetal bovine serum; HPL, human platelet lysate; Hplasma, human plasma).